Bacterial Magnetic Particles Research Articles

Molecular Mechanism of Magnet Formation in Bacteria.

Magnetic bacteria have an ability to synthesize intracellular ferromagnetic crystalline particles consisting of magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ) which occur within a specific size range (50–100 nm). Bacterial magnetic particles (BMPs) can be distinguished by the regular morphology and the presence of an thin organic membrane enveloping crystals from abiologically formed magnetite. The particle is the smallest magnetic crystal that has a regular morphology within the single domain size. Therefore, BMPs have an unfathomable amount of potential value for various technological applications not only scientific interests. However, the molecular and genetic mechanism of magnetite biomineralization is hardly understood although iron oxide formation occurs widely in many higher animals as well as microorganisms. In order to elucidate the molecular and genetic mechanisms of magnetite biomineralization, a magnetic bacterium Magnetospirillum sp. AMB-1, for which gene transfer and transposon mutagenesis techniques had been recently developed, has been used as a model organism. Several findings and information on the BMPs formation process have been obtained within this decade by means of studies with this model organism and its related one. Biomineralization mechanism and potential availability in biotechnology of bacterial magnets have been elucidated through molecular and genetic approach.

Two-dimensional analysis of proteins specific to the bacterial magnetic particle membrane from Magnetospirillum sp. AMB-1.

We report the identification of five proteins expressed specifically on the bacterial magnetic particle (BMP) membrane of Magnetospirillum sp. AMB-1. These proteins are major components of the BMP membrane. The molecular weights were determined to be 12.0, 16.0, 24.8, 35.6, and 66.2 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Of these five, the 16.0-kDa protein was the most abundant in the BMP membrane. Furthermore, the 16.0-kDa protein consisted of two components each of differing pI. The 35.6-kDa protein was the second most abundant protein of the five detected.

Production of luciferase-magnetic particle complex by recombinantMagnetospirillum sp. AMB-1

Luciferase-bacterial magnetic particle (BMP) complexes were produced by recombinant Magnetospirillum sp. AMB-1. We constructed plasmids pKML and pNELM, respectively, by fusing luc to the 5' and 3' terminal of magA, encoding an integral iron translocating protein situated in the BMP membrane, of AMB-1. In addition, we produced bifunctional active-fusion proteins on BMPs by using a plasmid pAcML. In this plasmid, acetate kinase and luciferase genes were fused to the N-terminus and the C-terminus of MagA, respectively. Bacterial magnetic particles isolated from transconjugants for pKML, pNELM and pAcML exhibited luciferase activity. Bacterial magnetic particles isolated from transconjugants for pAcML also exhibited acetate kinase activity. Fed-batch culture of pKML transconjugant yielded 2.6 mg BMPs per liter of culture, and 95% conversion of iron into magnetite was obtained, at a nitrate concentration of 1.4 mM. Continuous feeding of iron as ferric quinate significantly enhanced growth and total magnetic production. Final cell concentration of 1.8 x 10(9) cells/mL and 6 mg per liter of culture was obtained. Magnetite production by fed-batch culture of AMB-1 was about 3 times that obtained by batch culture. There were no significant differences in BMPs yield between recombinant AMB-1 cultivated by fed-batch culture and wild type of AMB-1.

Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1–10 3 ng/ml.

Biomagnetic nanoparticle formation and application

Magnetospirillum sp. AMB-1 is a bacterium that synthesizes intracellular particles of magnetite (Fe 3O 4). A magA gene required for the synthesis of bacterial magnetic particles (BMPs) was isolated from this strain and its gene product (MagA protein) was characterized as an iron transport protein. Intracellular localization analysis of the MagA protein using magA– Luc fusion proteins showed that MagA protein is localized on the surface of the lipid bilayer covering the BMPs. One particular BMP-associated protein, designated MpsA, was also purified from this strain and characterized. By the fusion-protein method using mpsA-luc genes, it was demonstrated that Mps-protein is preferentially partitioned onto the BMP membrane. Furthermore, application of BMPs to immunoassay is also described.

Isolation and Analysis of Surface Protein Gene from Bacterial Magnetic Particles

Isolation and Analysis of Surface Protein Gene from Bacterial Magnetic Particles

Magnetic Bacteria and Biomineralization

Magnetospirillum sp. AMB-1 is a magnetic bacterium which synthesizes intracellular particles of magnetite (Fe3O4). We isolated magA gene required for the synthesis of bacterial magnetic particles (BMPs) and it encodes a polypeptide which is an iron transport protein. Intracellular localization of the MagA protein was studied using magA - luc fusion proteins. The fusion protein was also detected on the surface of the lipid bilayer covering the BMPs. One particular BMP-associated protein designated “Mps” was also isolated and characterized. The localization analysis using mps-luc fusion protein showed that there is a significant preference for the magnetosome membrane during partitioning of the Mps and the Mps protein preferentially associates with the magnetosome membrane.

Magnetic control of bacterial magnetite-myosin conjugate movement on actin cables

The control of actin-myosin movement using a bacterial magnetic particle (BMP) which was synthesized intracellularly by magnetic bacteria was investigated. Myosin immobilized on the BMP ( myosin-BMP conjugate) was prepared with heterobifunctional reagents, Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3′-(2-pyridyldithio)-propionamido)hexanoate) and Sulfo-SMCC (sulfosuccinimidyl 4-( N-maleimidomethyl)cyclohexane-1-carboxylate). When a magnetic force was applied in the same direction as the myosin-BMP conjugate movement (negative magnetic force) at 22–23°C, the velocity increased from 2.0 μm s −1 to 7.5 μm s −1. When a magnetic force was applied in the opposite direction to the myosin-BMP conjugate movement ( positive magnetic force ), the velocity decreased from 10.7 μ s −1 to 0 μm s −1. These results indicate that the sliding force of the myosin-BMP conjugate caused by the actin-myosin interaction can be controlled using a neodymium-boron (Nd-B) magnet. It will be possible to develop a nano-sized actuator that can be controlled by the external magnetic force. This system could also be applicable to analysis of the dynamics of a single myosin molecule.

Magnetic Characterization of Bacterial Magnetic Particles

Mossbauer measurement, chemical analysis (ratio of Fe 2+ to total Fe content), analyses of the rotational hysteresis loss (Wr) and the magnetic viscosity coefficient (Sv) for bacterial magnetic particles covered with organic thin films (BMPs) were investigated and compared with the results for Fe 3 O 4 fine particles prepared by a chemical coprecipitation method. The calculated value of the saturation magnetization (Js) of BMPs was found to be 1.06x10.4 Wb m/kg (84.7 emu/g) using Mossbauer spectroscopy and chemical analysis. The Wr/Js versus H curve for BMPs had a stronger peak than that of Fe 3 O 4 fine particles (Sample C1). The rotational hysteresis integrals (Rh) for BMPs and Fe 3 O 4 fineparticles were 1.12 and 0.62, respectively. It is believed that they have different magnetization reversal mechanisms. The Sv value of BMPs at 127A/m was 50% smaller than that of the Fe 3 O 4 fine particles (C1). The diameter (Dact) of the activation volume (minimum volume for magnetization reversal) calculated from Sv, and the critical diameter (Dc) obtained by considering the superparamagnetic behavior for BMPs, were 39 and 22 nm, respectively. As the Fe 3 O 4 fine particles (C1) have smallerDact(31 nm) and Dc(18 nm), it may be concluded that BMPs is magnetically more stable than Fe 3 O 4 fine particles (C1).

Enhancement of magnetic particle production by nitrate and succinate fed-batch culture of Magnetospirillum sp. AMB-1

Magnetospirillum sp. AMB-1 is a magnetic bacterium, which is capable of growing under air atmosphere. This bacterium was employed to make bacterial magnetic particles (BMPs). AMB-1 only makes BMPs during logarithmic growth phase under anaerobic conditions. Since it requires nitrate as a nitrogen source, control of nitrate concentration in the medium was necessary. The fed-batch culture of AMB-1 was carried out by adding nitric acid and succinate as nitrogen and carbon source respectively. One liter of AMB-1 culture produced 0.34 g of dry cells and 4.5 mg of BMPs. BMP production by AMB-1 cultivated in the fed-batch culture was found to be seven times higher than that cultivated in the batch culture.

Chemiluminescence Enzyme Immunoassay Using Bacterial Magnetic Particles

A novel chemiluminescence enzyme immunoassay using bacterial magnetic particles (BMPs) has been developed for highly sensitive and rapid detection of immunoglobulin G. Antibody was immobilized onto BMPs using the heterobifunctional reagents sulfosuccinimidyl 6-[3'-(2-pyridyldithio) propionamido]hexanoate (sulfo-LC-SPDP) and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). For the highly sensitive immunoassay method using these BMPs, a good relationship was obtained between the luminescence intensity and mouse IgG concentration in the range of 1-10(5) fg/mL. Furthermore, in order to reduce assay time and to simplify operations, a rapid chemiluminescence enzyme immunoassay method has been developed. The rapid method was completed in 10 min. A linear relationship was obtained between the luminescence and mouse IgG concentration in the range of 10-1000 ng/mL.

磁性細菌ＡＭＢ‐１から分離した磁性微粒子の磁気的性質

The experimental results on Mossbauer measurement, chemical analysis (a ratio of Fe2+ to total Fe content), analyses of the rotational hysteresis loss (Wr) and the measurement of magnetic viscosity coefficient (Sv) for bacterial magnetic particles (BMP, particle diameter D=25-65 nm) were compared with those of Fe3O4 particles (D=18-45 nm) prepared by the chemical coprecipitated method. The value of saturation magnetization (Js) calculated from Mossbauer spectrum and the results of chemical analysis for BMP was 1.06×10-4 Wb m/kg. The curves of Wr/Ms versus H for BMP showed more sharp peaks than those of Fe3O4 particles. The rotational hysteresis integral (Rh) of BMP and Fe3O4 particles were 1.12 and 0.62, respectively. It was considered that they have different magnetization reversal mechanism. Sv value of BMP was 127 A/m, which was 50% smaller than that of Fe3O4 particles. The diameter (Dact) of activation volume (minimum unit volume of magnetization reversal) calculated from Sv and the critical diameter (Dc) obtained by considering the superparamagnetic behavior of BMP, were 39 and 22 nm, respectively. On the other hand, Fe3O4particles had smaller Dact (31nm) and Dc (18nm). It maybe concluded that the magnetic stability of BMP is better than that of Fe3O4 particles.

Iron-Regulated Expression and Membrane Localization of the MagA Protein in Magnetospirillum sp. Strain AMB-11

The magA gene from Magnetospirillum sp. strain AMB-1 is required for the synthesis of bacterial magnetic particles (BMPs). This gene has been cloned, sequenced and found to encode a protein which is homologous to the Escherichia coli potassium efflux membrane-binding protein, KefC. By using the firefly luciferase gene (luc) cloned downstream of the magA promoter, the effect of iron on regulation of magA expression was investigated, and transcription of magA was found to be enhanced by low concentrations of iron. Intracellular localization of the MagA protein was studied using magA-luc fusion proteins. The luc gene was cloned downstream of the magA hydrophilic C-terminal domain. Detection of luciferase activity in the cytoplasm, cell membrane, and magnetic particle membrane subcellular fractions confirmed that the MagA fusion protein was localized in the cell membrane. The fusion protein was also detected on the surface of the lipid bilayer covering the magnetic particles. These results suggest that MagA is a membrane-bound protein, the expression of which is enhanced at low iron concentrations.

Application of bacterial magnetic particles as novel DNA carriers for ballistic transformation of a marine cyanobacterium

Bacterial magnetite particles (BMPs) of 50 to 100nm diam were used as DNA carriers for the ballistic transformation of the marine cyanobacteriumSynechococcus. BMPs were bombarded into the cyanobacterial cells at several bombardment velocities using a particle gun. Successful transformation and gene expression were confirmed by Southern hybridization and CAT assay, respectively. The BMPs were also observed in the cyanobacterial cells by transmission electron microscopy. These results suggested that BMPs can be used as carriers for introducing DNA into bacterial cells.

Application of bacterial magnetic particles for highly selective mRNA recovery system

A novel mRNA detection system was developed using DNA immobilized on bacterial magnetic particles isolated from the magnetic bacterium Magnetospirillum sp. AMB-1 (Matsunaga et al., 1991). Immobilization of oligonucleotides was carried out using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). As a model system, we have attempted to detect bonito growth hormone mRNA from the bonito pituitary gland. Oligonucleotides corresponding to the 5'end of bonito GH mRNA was covalently immobilized onto bacterial magnetic particles. The particles were then incubated in the pituitary gland lysate, and recovered by Sm-Co magnet, followed by washing. cDNA synthesis was carried out using reverse transcriptase from mRNA recovered using a bacterial magnetic particles. The cDNA synthesized was analyzed by PCR. As a result, a unique band of bonito GH gene was detected. Furthermore, using recombinant Escherichia coli containing cloned bonito GH cDNA under downstream of lac promoter, the transcriptional level of cloned bGH gene was analyzed.

Highly sensitive detection of allergen using bacterial magnetic particles

A fluoroimmunoassay method using magnetic particles for the highly sensitive detection of allergen was developed. Fluorescein isothiocyanate (FITC)-conjugated IgE antibody was immobilized on bacterial magnetic particles using a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The decrease in fluorescence intensity on aggregation of FITC-labelled bacterial magnetic conjugated was determined by spectrofluorimetry. The relative flourescence intensity decreased with increasing allergen concentration. A linear relationship was obtained between the relative flourescence intensity and allergen in the range of 0.5–100 pg ml 0−1.

Detection and removal of Escherichia coli using fluorescein isothiocyanate conjugated monoclonal antibody immobilized on bacterial magnetic particles.

A novel fluoroimmunoassay method using bacterial magnetic particles for the highly sensitive detection of bacteria has been developed. Fluorescein isothiocyanate (FITC) conjugated monoclonal anti-Escherichia coli antibody was immobilized onto bacterial magnetic particles (BMPs) using a heterobifunctional reagent, N-succinimdyl 3-(2-pyridyldithio)propionate (SPDP). E. coli cells were reacted with FITC-antibody-BMP conjugates for 15 min in an inhomogeneous magnetic field which enhanced aggregation. The cell/BMP complexes sedimented, causing relative fluorescence intensity of the solution to decrease with increasing microbial cell concentration. A linear relationship was obtained between the relative fluorescence intensity and cell concentration in the range of 10(2)-10(6) cells/mL. Selectivity of this detection system was satisfactory. Monoclonal antibody immobilized on BMPs was also applied to the specific removal of E. coli from the bacterial suspension.

Magnetic Bacteria

Magnetic bacteria synthesize intracellular particles of magnetite, which are aligned in chains and enveloped by a membrane. A new species of magnetic bacterium was recently isolated from aerobic sediment from a pond in Tokyo, and is capable of growing aerobically to high cell densities. This strain may therefore be grown easily in mass culture. In such large-scale cultures a stationary phase is reached after four to five days, with a final cell concentration of 2×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">9</sup> cells/ml. The small size of the bacterial magnetic particles and the inhibition of aggregation by intact magnetosome membranes result in a large surface/volume ratio. When bacterial magnetic particles were prepared by ultrasonication, they were well dispersed in water. Phospholipids comprised 58% of the total lipid content, and phosphatidyl ethanolamine accounted for 50% of the total phos-pholipid content. A gene relating to bacterial magnetic particles was cloned in Escherichia coli and analyzed. A system for magnetic bacteria gene cloning was also developed.

Immunoassay method for the determination of immunoglobulin G using bacterial magnetic particles

We have developed a novel immunoassay method using bacterial magnetic particles for the determination of immunoglobulin G (IgG). Fluorescein isothiocyanate (FITC) conjugated anti IgG-bacterial magnetic particles were prepared. The fluorescence quenching caused by agglutination of FITC-anti IgG antibody-bacterial magnetic particle conjugates was measured by using a fluorescence spectrophotometer. The aggregates based on specific immunoreaction were separated by a gelatin solution. The aggregation of bacterial magnetic particle conjugates was enhanced by application of a magnetic field. The relative fluorescence intensity correlated linearly with a concentration of IgG in the range 0.5-100 ng/mL.

FLUOROIMMUNOASSAY FOR THE DETERMINATION OF ALLERGEN USING BACTERIAL MAGNETIC PARTICLES

We have developed a novel fluoroimmunoassay method using bacterial magnetic particles for the determination of allergen. Fluorescein isothiocyanate (FITC) conjugated IgE antibody was immobilized onto bacterial magnetic particles. The decrease of fluorescence intensity by aggregation of FITC-labeled bacterial magnetic particle conjugates was determined by a fluorescence spectrophotometer. The relative fluorescence intensity decreased with increasing allergen concentration. A linear relationship was obtained between the relative fluorescence intensity and allergen concentration in the range of 0.1 100ng/ml. This method may be carried our across a wider concentration range having a linear response than when artificial magnetite particles are used.